The clinical problem associated with patients who present with a suspicious thyroid nodule continues to place clinicians and patients in situations where decisions about the surgical approach need to be made with inadequate information. Although fine needle aspiration (FNA) biopsy of a thyroid nodule is very sensitive in the detection of malignancy, it is indeterminate or suspicious in 20-30% of cases. There are over 100,000 patients each year who present with a suspicious thyroid nodule in the United States. Terminologies commonly used in suspicious cytopathology reports include the following: follicular or Hürthle cell neoplasm, suspicious for papillary or follicular variant of papillary thyroid cancer, or cellular atypia. Because clinicians often cannot determine malignancy, either pre- or intra-operatively, patients with suspicious thyroid lesions cannot be optimally managed. This often results in two scenarios: 1) patients who ultimately have a benign lesion on final histopathology may be subjected to unnecessary surgery; 2) patients with a malignant thyroid nodule may need to undergo a second operation for completion thyroidectomy only after a diagnosis of cancer is rendered on permanent histological section. Thus, there is a need for a diagnostic test that can distinguish more effectively between malignant and non-malignant thyroid tumors, and that can provide guidance as to whether aggressive treatment, such as a total thyroidectomy, should be administered.
Telomerase is a ribonucleoprotein complex that stabilizes linear chromosomes (e.g. human chromosomes) by adding telomere sequence (TTAGGG) repeats to their ends. The protein component of this complex, the telomerase reverse transcriptase catalytic subunit (TERT), has been characterized in a variety of species. The human form of the protein is designated as hTERT (human telomerase reverse transcriptase). The wild type hTERT mRNA contains 16 exons. In addition, alternative splicing of RNA transcribed from the hTERT DNA has been observed. Seven alternative splice sites have been reported for hTERT, giving rise to splice variants that may include three deletions and four insertions. See, e.g., J P Venables (2004) Cancer Res 64, 7647-7654; Kilian et al, (1997) Hum Mol Genet 6, 2011-2019; Killin et al., U.S. Pat. No. 6,916,642). The splicing patterns are presented schematically in FIGS. 1A and 1B herein. There are several possible combinations of these alternative splice sites resulting in a large number of potential variant transcripts, but only a few have been confirmed (Hisatomi et al. (2003) Neoplasia 5, 193-197). The sequence of the wild type hTERT mRNA, as used herein, is represented by SEQ ID NO:1 (taken from U.S. Pat. No. 6,916,642). Variants of this sequence, including updated sequences, polymorphisms, allelic variants, or the like, are also included. The numbering of the sequence of SEQ ID NO:1 is used herein to indicate the location of the splice sites. The sequence of the polypeptide translated from SEQ ID NO:1 is represented by SEQ ID NO:2.
The four insertions and one deletion (β-deletion, 182 nt) generated by the alternative splices result in premature termination and non-functional proteins (Hisatomi et al. (2003) (supra)). The β-deletion, in which exons 7 and 8 are deleted, at nucleotides (nt) 2286-2468, gives rise to a reading frame-shift at nucleotide 2287, which is joined to nucleotide 2469, and a subsequent termination codon at nucleotide 2605. The hTERT protein translated from this alternatively spliced mRNA is thus truncated. The 182 nt deleted β sequence (sometimes referred to herein as the (β-deletion) is represented by SEQ ID NO:3; the protein sequence translated from it is inactive and is represented by SEQ ID NO:4. The translation product of an mRNA having the α-splice (36 bp deleted within the RT motif A, extending from nt 2131-2166) has been shown in cell culture studies to be a dominant negative inhibitor of telomerase activity (Wick et al. (1999) Gene 232, 97-106). The sequence of this α-deletion (sometimes referred to herein as the α-sequence) is represented by SEQ ID NO:5; the polypeptide translated from it is represented by SEQ ID NO:6. The γ-deletion (189 bp) has been identified in hepatocellular carcinoma cell lines and is also believed to be non-functional (Kilian et al, (1997) (supra)).
Telomerase enzyme activity has been reported by several groups to be regulated by posttranscriptional alternative splicing of hTERT (See, e.g., Colgin et al. (2000) Neoplasia 2, 426-432; Fan et al. (2005) Clin Cancer Res 11, 4331-4337). Furthermore, the patterns of hTERT alternative splice variants are known to vary in ovary, kidney, uterine and breast cancer, compared to corresponding adjacent normal tissues (See, e.g., Colgin et al. (2000) (supra); Ulaner et al. (1998) Cancer Res 58, 4168-4172; Ulaner et al. (2000) Int J Cancer 85, 330-335; Yokoyama et al. (2001) Mol Hum Reprod 7, 853-857). To our knowledge, no studies have reported differences between alternative splice variant patterns in benign and malignant tumors that originate from the same tissue type, or splice variant patterns that are more specific markers of malignant or benign disease than overall hTERT transcript levels. Comparable TERT alternative splicing patterns, including the α and the β deletions, have been characterized from vertebrate species other than human; the precise locations of the splice sites and the sequences of the wild type transcript are readily available to a skilled worker.